UNIVERSITY MALAYSIA SABAH

CONSERVATION BIOLOGY (HS03)

DIVERSITY MICROB LAB 2 EXAMINATION OF LIVING MICROORGANISM
NAME MATRIX NUMBER DATE OF LAB DEMOS NAME : NUR NADIAH BINTI KAMARUZAMAN : BS11110483 : 29 OCTOBER 2012 : HASRIN ROSSLEY : MOHD AFIFI AHMAD HAMDAN LECTURERS NAME : SIR HAIRUL HAFIZ MAHSOL : DR CHARLES S. VAIRAPPAN

SUBMISSION DATE : 05 NOVEMBER 2012

Objectives : 1. Prepare and observe wet mount slides and hanging-drop slides. 2. Distinguish between true motility and Brownian movement. 3. Use a phase-contrast microscope. 4. Explain how phase-contrast and dark field microscopy differ from bright field microscopy.

Introduction Brightfield microscopy is the most elementary form of microscope illumination techniques and is generally used with compound microscopes. The name "brightfield" is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as brightfield microscopes. This technique can be used to view fixed specimens or live cells. Since many organic specimens are transparent or opaque, staining is required to cause the contrast that allows them to be visible under the microscope. Different stains and staining techniques are used depending upon the type of specimen and cell structure being examined. For example, Fuchsin is used to stain smooth muscle cells. Methylene blue is used to stain cell nuclei. Gram stain is used on bacteria and gives rise to the name gram-negative or gram-positive bacteria based on the reaction of the bacteria to the stain. Dark field microscopy is a method which also creates contrast between the object and the surrounding field. As the name implies, the background is dark and the object is bright. An annular stop is also used for dark field, but the stop is now outside the field of view. Only light coming from the outside of the beam passes through the object and it cannot be seen directly. Only when light from the stop is deflected and deviated by the object can it be seen. This method also produces a great deal of glare and therefore the specimen often appears as a bright silhouette rather than as a bright object of which much detail can be determined. Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well. Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Amoebae look like vague outlines in bright field, but show a great deal of detail in phase. Most living microscopic organisms are much more obvious in phase contrast. Motility of microbes can be seen in two ways, either true motility or Brownian movement. Brownian motion is a phenomena whereby small particles suspended in a liquid tend to move in psuedo-random or stochastic paths through the liquid, even if the liquid in question is calm. It is the result of asymmetry in the kinetic impacts of molecules that make up the liquid. True motility requires a flagella and is a true form of motility it is also seen through rotations in spiral movements by the bacteria. True motility is only seen in live bacteria.

METHOD HANGING-DROP PROCEDURE The depression slide was obtained. A small amount of petroleum jelly was picked up using toothpick. A cover slip was picked up and carefully scraped the petroleum jelly with an edge of the cover slip to get a small rim of petroleum jelly. The step was repeated with other three edges. The cover slip was placed on paper towel. A drop of peppercorn infusion was transferred to the cover slip. A slide was placed over the drop and quickly inverted so that the drop is suspended. The drop was kept hanging. The slide was examined under low power by locating the edge of the drop and moving the slide so the edge of the drop crosses the center of the field. The light was reduced with iris diaphragm and focused. Different sizes, shapes and type of movement were observed. It was then switched to high-dry and observation was recorded. It was not focus down. When finished, the slide was cleaned and the procedure was repeated using new cover clip with culture of Bacillus. Observations were recorded. The oil was wiped from the objectives lens and the microscope was returned to its proper place. SPECIMEN FROM LAKE WATER. A drop of water from the lake was added onto a clean slide and covered with cover slip. The edges were sealed using petroleum jelly that was red in colour. The petroleum jelly made sure that the suspended fluid does not evaporated easily and quickly. The slide was observed under microscope and the observations made were recorded.

Result Refer to appendixes.

Discussion From this experiment, we observed algae, fungi, protozoa and bacteria. We observed different shapes of bacteria such as Bacillus or rod, Coccus and Spirillum. The main purpose was to observed the motility of organisms. We used different techniques in order to accomplish the objectives. First the wet mount technique. Mostly used for aquatic samples, living organisms and natural observations, wet mounts suspend specimens in fluids such as water, brine, glycerin and immersion oil. Although wet mounts can be used to prepare a significantly wide range of microscope slides, they provide a transitory window as the liquid will dehydrate and living specimens will die. This techniques is also time limited. Organisms such as protozoa may only live 30 minutes under a wet mount slide; applying petroleum jelly to the outer edges of the cover slip creates a seal that may extend the life of the slide up to a few days. In contrast, hanging drop techniques is more complex technique, but it allows for longer-term observation and more reliable observation of motility. The hanging drop technique is a method in which a drop of bacterial suspension, preferably in mid-logarithmic phase, is enclosed in an air-tight chamber prepared in a special depression slide (having a concave depression in the center) or assembled from modeling clay (plasticine) which is a soft, malleable, and non-hardening material available at toy or hobby stores. Upon inoculating a batch of culture medium, bacterial cells go through successive phases starting with a "lag" adaptation phase the length of which varies with type of organism and nature of the environment. During this phase cells may experience shock due to the change in environment. In addition, if cells were transferred from an old culture or from the refrigerator, their flagella may have deteriorated; therefore at this stage of growth examination for bacterial motility is not recommended. Bacteria then start dividing at the maximum rate, entering a "log' phase in which numbers increase logarithmically (exponentially). Young actively dividing cells demonstrate the best motility at the log phase. There are two type of motility in microorganism. Its either true motility or Brownian movement. True motility requires a flagella and is a true form of motility it is also seen through rotations in spiral movements by the bacteria. True motility is only seen in live bacteria. While Brownian movement is not a true form of motility and is simply the result of vibrating particles that make the bacteria seem like they are moving on their own. This usually occurs in dead bacteria.

Phase contrast microscopy is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well. Phase contrast is a widely used technique that shows differences in refractive index as difference in contrast. Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Amoebae look like vague outlines in bright field, but show a great deal of detail in phase. Most living microscopic organisms are much more obvious in phase contrast. To use phase contrast microscopy, the light path must be aligned. An element in the condenser is aligned with an element in a specialized phase contrast lens. This usually involves sliding a component into the light path or rotating a condenser turret. The elements are either lined up in a fixed position or are adjusted by the observer until the phase effect is optimized. Generally, more light is needed for phase contrast than for corresponding bright field viewing, since the technique is based on a diminishment of brightness of most objects. The ring in the objective has special optical properties: it first of all reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about a quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image. Phase contrast and darkfield microscopy differ from brightfield microscopy. Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light. The most common use of the microscope involves the use of an organism mounted to a glass microscope slide. But, darkfield microscopy is a technique for improving the contrast of unstained, transparent specimens. Darkfield illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (un-scattered) light entering the image plane, collecting only the light scattered by the sample. Darkfield can dramatically improve image contrastespecially of transparent objectswhile requiring little equipment setup or sample preparation. However, the technique does suffer from low light intensity in final image of many biological samples, and continues to be affected by low apparent resolution.

Conclusion In a nutshell, this experiment gave us diverse benefits. We finally are able to prepare and observe wet mount sides and hanging-drop slides. Next, we can distinguish the movement of bacteria whether it is Brownian movement or true motility. We had also finally master the way to use the phase-contrast microscope wisely and last but not least, we can now explain briefly the difference between phase contrast and darkfield microscopy with brightfield microscopy.

Questions 1. Yes. The difference is Brownian movement is that first of all it is not a true form of motility and is simply the result of vibrating particles that make the bacteria seem like they are moving on their own. This usually occurs in dead bacteria. True motility is movement of the cell by appendages. The cells move to and fro and the movement is more obvious. True motility is movement in a specific direction. Flagella is very visible. 2. There are a few practical values in those techniques. The hanging drop and wet mount techniques allow for observation of living organisms. The wet mount tend to dry out quickly under the heat of the microscope light; it is simpler to perform than the wet mount, but it is useful for short-term observation only. The hanging drop is a more complex technique, but it allows for longer-term observation and more reliable observation of motility. These techniques are usually performed without the addition of any stains; therefore, the organisms can be difficult to see. Reduce the illumination on your microscope as much as you can while still allowing yourself enough light to observe the organism. 3. Hanging drop dry out slower, hence we get better view of true motility of the organisms. 4. The human eye is only sensitive to amplitude (intensity) and wavelength (color), which are observed in a normal bright field microscope. Small, transparent objects like a cell do not change these parameters much, but due to their different refractive index from the surrounding medium, they slow down the light that passes through them. The light gets diffracted and has a phase change of approx. 1/4th of the wavelength (depends on the object thickness). Phase contrast microscopes have two rings, one that provides a hollow cone of light that illuminates the specimen and a second (so called phase plate) which lets the unaltered light pass through a thinner part and the bent light through a thicker part .This introduces another relative phase

shift of 1/4, causing a net phase shift of 1/2 of the wavelength. Now this results in destructive interference, resulting in a dark object on a bright background. 5. So that the cover slip adheres to the slide and to keep the water in the depression and hence reduce rate of evaporation. 6. Hay infusion. This is because the bacteria was not killed and just left to live about. Instead they are just contained in the boundary of cover slip and can move freely and hence their motility can be observe better. Critical Thinking 1. In a wet mount the microorganisms are hard to see because they blend in with their surroundings or the liquid they are grown in. An example of this would be in a hay broth where the water is cloudy and the microorganisms are transparent. Plus, they move quickly and is hard to bring into focus.

2. Eukaryotic Cell Nucleus: Number of chromosomes: Cell Type: True Membrane bound Nucleus: Example: Telomeres: Present More than one Prokaryotic Cell Absent One--but not true chromosome: Plasmids Unicellular Absent

Multicellular Present

Animals and Plants Present (Linear DNA)

Bacteria and Archaea Circular DNA doesn't need telemeres Partial, undirectional transfers DNA Absent

Genetic Recombination: Lysosomes and peroxisomes:

Mitosis and fusion of gametes Present

Eukaryotic Cell Microtubules: Endoplasmic reticulum: Mitochondria: Cytoskeleton: DNA wrapping on proteins.: Ribosomes: Golgi apparatus: Mitosis: Chloroplasts: Present Present

Prokaryotic Cell Absent or rare Absent

Present Present Yes

Absent May be absent No

larger Present Yes Present (in plants)

smaller Absent No---but has binary fission Absent; chlorophyll scattered in the cytoplasm Submicroscopic in size, composed of only one fiber

Flagella:

Microscopic in size; membrane bound; usually arranged as nine doublets surrounding two singlets Selective

Permeability of Nuclear Membrane: Plasma membrane with steriod: Cell wall:

not present

Yes

Usually no

Only in plant cells (chemically simpler) 10-100um

Usually chemically complexed

Cell size:

1-10um

3. Oil immersion would make things worse because it would refract the light even more considering that the refraction index of the oil immersion and the slide [depression slide+drop] are different.

4. Protozoans (e.g. Paramecium sp) come from rehydrated cysts. Bacteria can come from bacterial cysts or endospores. Fungi and water molds come from spores. Animals (e.g. rotifers, worms, daphnia) come from eggs or cysts.

Reference 1. Neil A. Campbell, Jane B. Reece, 2005. Biology 7th Edition. Pearson Education. New York. 2. Prescott, Harley, Klein,2005. Microbiology 6th Edition. Mc Graw Hill. New York.